Angiogenesis is a mechanism in which new blood vessels are generated from existing blood vessels by growth, division, migration, and the like, of an endothelial cell, plays an important roll in normal growth processes including wound healing or female menstrual cycle (Risau, Nature, 386:671, 1997), and moreover, abnormally excessive angiogenesis is known to play a crucial role in diseases such as tumor growth and metastasis, age-related macular degeneration (ARMD), diabetic retinopathy, psoriasis, rheumatoid arthritis and chronic inflammation (Carmeliet and Jain, Nature, 407:249, 2000).
Hypothesis that tumor growth and metastasis are angiogenesis dependent, and therefore, a therapy focusing on anti-angiogenesis could be a new therapeutic agent for solid tumors was raised by Dr. J. Folkman in 1971. After that, research into a technology relating to inhibition of excessive angiogenesis mechanisms has attracted attention of many researchers (Ferrara and Kerbel, Nature, 438:967, 2005). A progressing aspect of the angiogenesis is determined by comprehensive balance of angiogenesis inducers and angiogenesis inhibitors, and is progressed by complex and multi-step sequential processes. In detail, various angiogenesis inducers including vascular endothelial growth factor (VEGF) secreted by tumor or injured tissues are bound to corresponding receptors of existing peripheral vascular endothelial cells to activate vascular endothelial cells, which increase permeability of vascular endothelial cells, and to secret protease such as matrix metalloproteinase (MMP), which decomposes basement membrane and extracellular matrix surrounding vascular endothelial cells, such that the vascular endothelial cells escape from existing capillaries and migrate/proliferate toward the tissue secreting angiogenesis inducer. The migrated and proliferated vascular endothelial cells form an intravascular tube structure, and finally, pericyte which is a structural support of the vascular endothelial cell is introduced to achieve stable and mature blood vessel formation.
As described above, it was found that signaling of VEGF and a VEGF receptor (VEGFR) bound to the VEGF is suppressed to ultimately inhibit angiogenesis, thereby obtaining therapeutic effects on various diseases age-related macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis and chronic inflammation, including growth and metastasis of tumor, and thus, development of various drugs capable of inhibiting VEGF activity has been ongoing.
Specifically, VEGF forms protein separation and purification and cDNA cloning by Dr. N. Ferrara group from Genentech in 1989 (Leung et al., Science, 246:1306, 1989). It is known so far that VEGF which is also referred to as VEGF-A has four isotypes (VEGF121, VEGF165, VEGF189, and VEGF206), and it is reported that among the four isotypes, VEGF165 is the most abundant in all human tissues except for placenta (Tisher et al., J. Biol. Chem., 266:11947, 1991). It is known that VEGF is bound to receptors VEGFR-1 and VEGFR-2/KDR with significantly high affinity; however, signal of VEGF is mainly transferred through VEGFR-2 to induce mechanisms related to angiogenesis such as proliferation, migration, and the like, of vascular endothelial cells. Due to the above-described reasons, VEGF and VEGFR-2 become main targets for inhibiting angiogenesis mechanism induced by VEGF, and a number of theses deal with VEGF and VEGFR-2 (Ellis and Hicklin, Nature Rev. Cancer, 8:579, 2008; Youssoufian et al., Clin. Cancer Res., 13:5544s, 2007).
For example, Avastin (bevacizumab, Genentech) is a humanized antibody targeting VEGF-A (Ferrara et al., Biochem. Biophy. Res. Comm., 333:328, 2005), which has received US FDA approval on treatment for metastatic colorectal cancer in 2004, non-small cell lung cancer in 2006, and Her-2 negative metastatic breast cancer in 2008, respectively, and is approved to treat Glioblastoma mutiforme (GBM), and renal cancer. Currently, clinical trials on a variety of solid tumors are ongoing in order to expand indications. In addition, Lucentis which was developed in the same company, is an antibody prepared by cutting Fab fragments only from Avastin for good permeability of Lucentis when Lucentis is injected into retina in order to inhibit excessive angiogenesis around macula which is a main aspect of senile macular degeneration (Eter et al, Biodrgus, 20:167, 2006), and as a therapeutic agent for wet age-related macular degeneration (wet-ARMD), which has received US FDA approval in 2006.
As another antibody for treatment targeting VEGF, there is VEGF-trap manufactured by Regeneron (Holash et al., PNAS, 99:11393, 2002). VEGF-trap is a soluble decoy receptor in a form in which second immunoglobulin domain of VEGFR-1 and third immunoglobulin domain of VEGFR-2 are fused to human Fc, which has not received U.S. FDA approval yet, but has been ongoing in phase III stage for metastatic breast cancer, metastatic lung cancer, metastatic colorectal cancer, hormone refractory prostate cancer, and the like.
Meanwhile, examples of anti-angiogenesis antibodies targeting VEGFR-2 which is a receptor of VEGF include IMC-1121B (EP 1916001A2) manufactured by Imclone company, CDP-791 (PCT/GB02/04619) manufactured by UCB company, Tanibirumab (TTAC-0001) (WO2008/153237) developed by the present inventors and has been in a clinical trial, and the like.
IMC-1121B is a monoclonal antibody selected from a fully human Fab library, which has been ongoing in Phase III stage for metastatic breast cancer, and was entered in Phase III stage for stomach cancer in 2010. CDP-791 manufactured by UCB is a humanized antibody, which has been ongoing in phase II stage for non-small cell lung cancer in PEGylated Di-Fab form. Since this antibody does not have Fc, antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity may not be expected.
Lastly, Tanibirumab (TTAC-0001) developed by the present inventors is a monoclonal antibody selected from a fully human ScFv library, and is the only antibody having reactivity with flk-1 of mouse and rat origin (VEGFR-2 homologue) while simultaneously targeting VEGFR-2, which is one of important distinguishable features from IMC-1121B manufactured by Imclone (WO2008/153237). In particular, cross-species cross reactivity exhibited by Tanibirumab is possible to make a research into animal disease model to carry on future development of anti-cancer agent for specific cancer by stages, which makes related researches easier.
As described above, researches targeting VEGF and VEGFR-2 have been dramatically developed for last five years, and a number of therapeutic agents are developed by market and clinical studies.
Meanwhile, cells differentiated into Tip cell by VEGF/VEGFR-2 signaling strongly express DLL4 and are bound to Notch1 receptor present in surrounding cells, and cells in which Notch1 signaling pathway is activated are differentiated into stalk cellIs to form normal blood vessel tube structure, which proves that DLL4/Notch1 signaling pathway is one of the most important mechanisms for VEGF/VEGFR-2 path and angiogenesis (Dufraine et al., Oncogene, 27:5132-5137, 2008).
It is known so far that DLL4 is one of ligands to a Notch receptor, and there are four kinds of Notch receptors (Notch 1 to 4) and five kinds of Notch ligands (Jagged-1, Jagged-2, DLL1, DLL3, and DLL4) in mammals. Notch signaling pathway is initiated by binding a Notch ligand of one cell to a Notch receptor of other cell, and is necessarily activated only by direct interaction between different cells (Bray SJ, Nat Rev Mol Cell Biol., 7(9):678, 2006).
When the Notch ligand is bound to the Notch receptor, an ADAM metalloprotease is firstly activated to cleave a cellular membrane outer proximal site of the Notch receptor, and then a gamma-secretase complex is activated to cleave a cellular membrane inner proximal site of the Notch receptor, such that Notch Intracellular Domain (NICD) is isolated and migrates into the nucleus. NICD is bound to an RBPJ/CSL transcription factor to induce expression of Notch target genes such as basic helix-loop-helix proteins including Hes and Hey. The Notch signaling pathway determines proliferation/differentiation/apoptosis in accordance with the situation of corresponding cells, and plays an important role in maintenance of normal stem cells and cancer stem cells.
Basically, all Notch receptors are capable of being bound to all Notch ligands; however, combinations of various bindings are selectively controlled in microenvironments of the corresponding cells. For example, DLL4 is strongly expressed on angiogenesis endothelial cells during a fetal development process, and is bound to Notch1 and Notch4 which are expressed in peripheral endothelial cells; however, DLL4-Notch1 binding is the most important in an exclusive way (Yan M, Vasc Cell, 2011), and angiogenesis progresses through the DLL4-Notch1 binding. The above-description is well found by gene deficiency test, and the like (Duarte et al., Genes Dev, 2004; Gale et al., PNAS, 2004; Krebs et al., Genes Dev, 2004).
Therefore, when the DLL4-Notch1 binding is suppressed, angiogenesis may be inhibited, and therefore, various diseases such as tumor, and the like, are capable of being treated. It has been already proven that when VEGF is inhibited by using Avastin (bevacizumab), and the like, in cancer treatment, angiogenesis is inhibited to decrease perfusion of the tumor, and a tumor size is decreased. Meanwhile, when binding with Notch1 expressed in peripheral cells while targeting DLL4 is inhibited, blood vessels are abnormally and largely generated (hypersprouting), but do not achieve complete function, which decreases perfusion of non-functional tumor, and as a result, the tumor size is reduced (Thurston et al, Nat Rev Cancer, 7(5):327, 2007).
Interestingly, when an antibody inhibiting VEGF and DLL4 is administered in xenograft animal experiments using several cancer cell lines performed in Genentech's research team, growth of the cancer is much strongly suppressed, as compared to a case in which an antibody inhibiting VEGF and an antibody inhibiting DLL4 are separately administered, respectively (Ridgway et al., Nature, 444(7122):1083, 2006). It suggests that signaling by DLL4/Notch1 path is not simply activated by VEGF/VEGFR-2 path, and various angiogenesis-related diseases such as tumor, and the like, are capable of being effectively treated by simultaneously inhibiting signalings by two paths.
In addition, it was found that DLL4 inhibition has an effect on both of a tumor being sensitive to VEGF/VEGFR-2 path inhibitor and a tumor being resistant to VEGF/VEGFR-2 path inhibitor (Ridgway et al., Nature., 444(7122):1083, 2006; Noguera-Troise et al., Nature., 444(7122):1032. 2006), which provides a significantly important clue to overcome resistance which currently and frequently occurs when drugs such as Avastin blocking VEGF are administered (including two cases of an intrinsic resistance in which Avastin is not effective from the beginning and acquired resistance in which an efficacy of Avastin is gradually falling over time).
Further, it was found from Oncomed's research team that DLL4 inhibition directly reduces frequency of cancer stem cells in tumor and inhibits tumor growth (Hoey et al., Cell Stem Cell., 2009), which suggests that DLL4 inhibition is possible to essentially block recurrence of cancer. Finally, resistance to anti-cancer chemotherapy and antibody therapeutic agents such as Herceptin, and the like, that are currently used for cancer treatment has a lot of relevance to the Notch signaling pathway and inhibition of DLL4/Notch1 path is also possible to overcome resistance of the anti-cancer chemotherapy and the antibody therapeutic agents such as Herceptin, and the like (Wang et al., Biochim Biophys Acta., 1806(2):258, 2010).
As described above, various angiogenesis-related diseases such as tumor, and the like, are capable of being effectively treated by simultaneously inhibiting signalings by two paths of VEGF/VEGFR-2 and DLL4/Notch 1. However, the development of drugs that are effective for this has not been made yet, and therefore, relevant development is urgently required.